Modified vaccinia Ankara (MVA) is a highly attenuated member of the genus Orthopoxvirus in the family of Poxviridae. Poxviruses engineered to express foreign genes are established tools for target protein synthesis and vaccine development in biomedical research. Their favorable characteristics include a large packaging capacity for recombinant DNA, precise virus-specific control of target gene expression, lack of persistence or genomic integration in the host, high immunogenicity as vaccine, and ease of vector and vaccine production.
MVA arose as an alternative to vaccinia virus (VV) smallpox vaccine after safety concerns instigated the development of viruses that are replication-defective in human cells. After more than 570 passages in chicken embryo fibroblasts MVA had lost the broad cellular host range of VV, being unable to effectively grow in many cells of mammalian origin, including human cells. MVA has been used for the primary smallpox vaccination of more than 100,000 people without serious problems and was considered avirulent after testing in laboratory animals.
For most purposes the generation of MVA vectors requires a single genomic insertion from a plasmid that carries one or two recombinant genes being placed under control of a VV-specific promoter. The sites of naturally occurring deletions within the MVA genome or the gene loci encoding the VV proteins thymidine kinase or hemagglutinin serve as sites for the insertion of recombinant gene sequences.
Much previous research has been dedicated to the development of MVA candidate recombinant vaccines against multiple virus infections of humans, including those causing AIDS, influenza, early childhood respiratory diseases, measles, Japanese encephalitis, dengue fever or malaria. As an effective vaccine against AIDS is urgently needed, recombinant MVA producing immunodeficiency virus antigens are among the first vector viruses to be evaluated as candidate vaccines in humans.
Viruses are often unstable outside their native environments, which can vary considerably among cell compartments and extracellular fluids. If certain conditions are not maintained, purified viruses may not function properly or remain soluble. Furthermore, virus titer can be affected by proteolysis, aggregation and suboptimal buffer conditions. Purified viruses for use in vaccination, for example, often need to be stored for extended periods of time while retaining their original structural integrity and/or activity. The extent of storage ‘shelf life’ can vary from a few weeks to more than a year and is dependent on the nature of the virus and the storage conditions used.
To ensure stability, therapeutic viral formulations are generally supplied either as lyophilized material to be dissolved just before use in a separately packaged water soluble diluent; however, this process increases manufacturing costs, and involves an increased risk of improper administration as the lyophilized protein needs to be dissolved just prior to use and, usually, a loss in infectivity titer is seen after the lyophilization process. Alternatively, therapeutic viral formulations may be supplied as solutions containing additives for improving stability. For example, additives such as free amino acids (e.g., leucine, tryptophan, serine, arginine and histidine) useful in formulating protein solutions have been proposed. Some viral formulations currently available on the market contain a protein as a stabilizer. Human serum albumin (HSA) or purified gelatin can be used to suppress chemical and physical changes in viral solutions. However, the addition of these proteins involves a complicated process for removing viral contamination. Furthermore, they can produce strong anaphylactic responses which limits their use.
Liquid viral formulations are commonly stored as frozen solids. Conventional cryopreservation utilizes a range of additives to promote vitrification. Vitrification is a process of converting a material into a glass-like amorphous solid which is free from any crystalline structure, either by the quick removal or addition of heat, or by mixing with an additive. Solidification of a vitreous solid occurs at the glass transition temperature (which is lower than the melting temperature, Tm, due to supercooling). Additives used in cryobiology or produced naturally by organisms living in polar regions are called cryoprotectants. Conventional cryoprotection focuses on achieving a solid state which is most favorable for the long-term storage of biological materials, i.e., on the transition from a liquid to a solid state.
However, it is the surprising finding of this study that once a solid frozen state is achieved, viral compositions may be unstable.